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as proteomics techniques. A good example of this is the “two-
hybrid system,” directed at studying gene product interactions (
see
Chapter
18
)
. Within this so-called wide genomics group, we can
include other approaches like the analysis of gene tagged proteins
[
27
] and the
in silico
analysis of the gene sequence [
28
,
29
]. Finally,
structural proteomics, aiming at generating protein 3D structures
after
in silico
, crystallographic, or spectroscopic analysis, could also
be considered within proteomics. Unfortunately we could not, but
also we did not intend it, to cover in detail all of them in this topic,
so we have focused on MS-based proteomics techniques, leaving
out methods of wide genomics or structural proteomics.
The workfl ow of a standard MS-based proteomics experiment
includes all or most of the following steps: experimental design,
sampling, tissue/cell or organelle preparation, protein extraction/
fractionation/purifi cation, labeling/modifi cation, separation, MS
analysis, protein identifi cation, statistical analysis of data, validation
of identifi cation, protein inference, quantifi cation, and data analy-
sis, management and storage. The different strategies for a pro-
teomics experiment are the result of different protocol combinations
in a specifi c sequence, and can be named and grouped into differ-
ent categories.
MS analysis can be performed with either proteins (protein
centric) or peptides resulting from its protease (usually trypsin, but
may be others if lack of trypsin cleavage sites,
see
Chapter
14
)
digestion (peptide centric), being them called as top down (pro-
teins) or bottom up (peptides) proteomics, respectively. We left
out of this topic the top-down strategy, but readers are referred to
some publications [
30
,
31
]. In both top and bottom approaches,
MS analysis can be carried out with total protein extracts (in top
down) or digested peptide products (in bottom up) Alternatively,
it can be made after a protein separation step, in most of the cases
using electrophoresis (one or two dimensional gel electrophoresis)
and, to a lesser extent, conventional liquid chromatography, known
as gel-based or LC-based (gel-free) techniques, respectively. The
simplest technique in terms of sample manipulation and number of
steps is the so called MudPIT, in which peptides derived from total
protein extracts digestion are subjected to MS analysis after LC
(nano or micro) separation [
31
]. In terms of proteome coverage
and number of proteins identifi ed, a combination of 1-DE, protein
digestion of specifi c bands or group of bands, coupled to on line
nLC-ESI-MS, has proved to be the most powerful technique (
see
Chapters
21
,
25
,
27
,
29
)
.
Depending on the quantifi cation strategy used (MS-based
quantifi cation), we may refer to relative quantitation based on label
(DIGE, ICAT, iTRAQ, SILAC) and label-free (peak area or
spectral counting) strategies, being them illustrated by Chapters
11
,
12
,
32
(label), and
13
,
20
,
22
(label-free). Some of the labeling
techniques, like ICAT, have been used to monitor oxidoreduction
of protein thiols processes (
see
Chapter
47
)
. Recently, the absolute
